A novel strategy for screening bioavailable quality markers of traditional Chinese medicine by integrating intestinal absorption and network pharmacology: Application to Wu Ji Bai Feng Pill

BackgroundMajor components are often used as marker compounds for quality control of traditional Chinese medicines (TCMs). However, these compounds may not necessarily bioavailable and active in vivo, thereby, failing to control the “quality”.PurposeThe purpose of this paper is to develop a novel strategy integrating absorption and activity deduced from network pharmacology to identify more reasonable markers for quality control of TCM formulas using Wu Ji Bai Feng Pill (WJBFP) as an example.Study DesignHuman Caucasian colon adenocarcinoma (Caco-2) cell transport studies and a bioavailability-enhanced network pharmacological approach were integrated to identify better phytochemical markers for quality control.MethodsThe absorption of multiple components in WJBFP was evaluated by a Caco-2 cell culture model. Nine databases were used to identify potential targets in the network pharmacology analysis. Cytoscape 3.7.2 was employed for the network data integration, visualization, and centrality analysis. Molecular docking was carried out to investigate the binding affinity of the identified markers to their candidate targets.ResultsThe apparent permeability coefficient (Papp) and efflux ratio (ER) of 66 compounds were determined. Five hundred and two putative targets and 187 primary dysmenorrhea (PD) related targets were identified. Twenty-two candidate targets interacting with 20 potential active compounds were screened with the putative PD related targets intersection network using Degree Centrality (DC) ranking. By integrating absorption, 16 candidate targets interacting with 8 potential active compounds were identified. Besides, 53 compounds hitting candidate targets were divided into two classes according to their DC values. Then each of the two classes of DC was stratified into two groups based on the Papp for a total of four classes. Finally, five compounds belonging to Class 1 with higher DC and higher Papp, formononetin, ferulic acid, isoliquiritigenin, neocryptotanshinone and senkyunolide A, were identified as potential bioavailable phytochemical markers for the quality control of WJBFP against PD. Furthermore, molecular docking analysis validated the interplay between candidate targets and marker ingredients.ConclusionA novel strategy combining intestinal absorption with network pharmacology analysis was successfully established to identify bioavailable and bioactive markers for quality control of WJBFP against PD.     

Higenamine alleviates allergic rhinitis by activating AKT1 and suppressing the EGFR/JAK2/c-JUN signaling

BackgroundAllergic rhinitis (AR) is an inflammatory, immunoglobulin E (IgE)-mediated disease characterized by the typical symptoms of sneezing, rhinorrhea, nasal itching, and congestion. Higenamine (HG) is a plant-based alkaloid, possesses a wide range of activities, including vascular and tracheal relaxation, antioxidative, antiapoptotic, anti-inflammatory, and immunomodulatory activities. So far, the effect and the underlying mechanism of HG on AR have not been studied.Hypothesis/PurposeThe purpose of this study was to evaluate the effects of HG on AR and investigate its underlying mechanism.MethodsThe effects of HG on AR were evaluated in an ovalbumin-induced AR mouse model. Network pharmacology-based methods such as target prediction, protein-protein interaction (PPI) network analysis, pathway analysis, and molecular docking were used to identify the likely HG targets. Finally, we validated the mechanism of action of HG through its effects on these targets in human nasal epithelial cells (HNEpCs).ResultsOral administration of 30, 60, and 120 mg/kg HG significantly alleviated rubbing and sneezing in AR mice and attenuated histopathological changes in the lung and nasal tissues. Additionally, HG reduced the levels of IgE, histamine, and IL-4 in the serum of AR mice, and regulated imbalance in Th1/Th2 cells. Using network pharmacology-based methods, we identified 29 HG targets related to AR. These targets are mainly involved in the PD-L1, relaxin, estrogen, HIF-1, Th1 and Th2 cell differentiation, T cell receptor, and the Th17 cell differentiation signaling pathways. Molecular docking showed that HG may well be suited to the receptor binding pockets of key target AKT1, EGFR, c-Jun, NOS2, and JAK2. In HNEpCs, HG inhibited the histamine-induced mRNA expression and secretion of interleukin (IL)-6, and IL-8, as well as the expression of MUC5AC and the phosphorylation of NF-κB. Moreover, HG affected the changes of AKT1, EGFR, c-Jun, iNOS, and JAK2 induced by histamine.ConclusionOverall, our results suggest that HG may alleviate AR by activating AKT1 and suppressing the EGFR/JAK2/c-JUN signaling. HG, therefore, has great potential as a therapeutic agent for the treatment of AR.     

Protective immunity of <Emphasis Type="Italic">E. coli</Emphasis>-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.     

Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen

Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD₅₀), which was abrogated with the increase of viral dose (40 LD₅₀). The pcTPANS1 also induced activation of CD4⁺ and CD8⁺ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8⁺ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4⁺ cells. Depletion of CD4⁺ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4⁺ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4⁺ T cells and the humoral immunity.     

Investigating the medicinal potential,material basis and mechanism of Polygoni Orientalis Fructus based on multi-technology integrated network pharmacology

BackgroundPolygoni Orientalis Fructus (POF) refers to the dried ripe fruit of Polygonum orientale L. which has a long historical application in clinic for treatment of various conditions in China. However, its chemical constituents, pharmacological effects and their coupled correlation have not been intensively investigated.PurposeIn present work, we aimed to elucidate the medicinal material basis, optimum indication and corresponding therapeutic mechanism of POF.MethodsThe main phytochemical ingredients in POF were characterized by liquid chromatography-mass spectrometry (LC-MS) analysis. The optimum medicinal potential and corresponding molecular mechanism of POF were deduced based on integrated statistic pattern recognition and network pharmacology. The deduced pharmacologic efficacy and mechanism of POF were further validated through in vitro study in free-fatty acid (FFA)-induced LO₂ cells.ResultsTotal 30 main phytochemical ingredients were identified in POF in which 18 ingredients were screened to yield 277 potential targets. Based on analyzing the quantitative data matrix of drug-disease targets by statistic pattern recognition, non-alcoholic fatty liver disease (NAFLD) was screened as the optimum indication of POF from 23 candidate diseases. Promising action targets (PPARG, IL6, TNF, IL1B, IKBKB, RELA, etc.) and signaling pathways (AMPK signaling pathway, NF-κB signaling pathway, etc.) were screened and refined to elucidate the therapeutic mechanism of POF against NAFLD based on network pharmacology. In vitro study demonstrated that POF effectively alleviated FFA-induced steatosis, oxidative stress, mitochondrial dysfunction and inflammation, and these beneficial effects were attributed to the activation of AMPK signaling pathway and suppression of NF-κB signaling pathway.ConclusionPOF could be exploited as a promising phytotherapy in the treatment of NAFLD.     

Antibodies against dengue virus nonstructural protein-1 induce heme oxygenase-1 via a redox-dependent pathway in human endothelial cells

Heme oxygenase (HO)-1, the inducible isoform of the first and rate-limiting enzyme of heme degradation, affords anti-inflammatory protection via its cell-type-specific effects in endothelial cells (ECs). In dengue hemorrhagic fever (DHF), which is the life-threatening form of dengue virus (DV) infection, endothelial interactions of cross-reactive antibodies against the DV nonstructural glycoprotein-1 (NS1) are associated with endothelial dysfunction. In this study, we investigated whether anti-NS1 antibodies might regulate HO-1 gene expression in human ECs. Serum from DHF patients with high anti-NS1 titers and a monoclonal anti-NS1 antibody upregulated HO-1 gene expression in human umbilical vein ECs, which was blocked by purified NS1 antigen. Immunoprecipitation studies showed that anti-NS1 antibodies specifically bound to the oxidoreductase protein disulfide isomerase (PDI) on ECs. Moreover, anti-NS1-mediated HO-1 induction was reduced by inhibition of PDI enzyme activity. Reactive oxygen species, which were generated by NADPH oxidase and in turn activated the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, were involved in this upregulation of HO-1 gene expression. Finally, apoptosis of ECs caused by anti-NS1 antibodies was increased by pharmacological inhibition of HO-1 enzyme activity. In conclusion, HO-1 gene expression is upregulated by anti-NS1 antibodies via activation of a redox-dependent PDI/PI3K/Akt-mediated pathway in human ECs.     

A combined network pharmacology and molecular biology approach to investigate the active ingredients and potential mechanisms of mulberry (Morus alba L.) leaf on obesity

BackgroundAs one of traditional Chinese medicine, mulberry leaf is abundant in diverse active ingredients and widely used for the treatment of metabolic disease and its complications. However, there are a few of reports on its application in the prevention and treatment of obesity. And the molecular mechanism on the anti-obesity of mulberry leaf are unknown till now.PurposeThe present study aimed to evaluate the potential ingredients and targets of mulberry leaf and uncover the anti-obesity mechanisms by using the network pharmacology tactics and verify its effect by biological experiments.Study designActive ingredients and key targets of mulberry leaf, genes related to obesity were screened through public database. Based on the results of network pharmacology, the flavonoids-enriched fraction of mulberry leaf (MLF) was extracted and composition of this fraction was identified. After that, HepG2 cells model of lipid accumulation was established for verifying the effect of MLF and related mechanisms.ResultsA total of 37 active ingredients in mulberry leaf, 192 predicted biological targets and 8813 obesity-related targets were determined, of which 180 overlapping targets might have obvious curative effects on obesity. The networks showed that mulberry leaf might play a role through key targets, such as AKT, MAPK and IL-6, and regulated PI3K-Akt signaling pathway. Based on HPLC-ESI-QQQ-MS analysis, 13 constituents of MLF were identified, including 9 flavonoids. Furthermore, HepG2 cells model of lipid accumulation was established. The results indicated that MLF treatment could down-regulate the secretion of inflammatory cytokines, as well as clearly inhibited lipid droplets formation and alleviated TC, TG, HDL-C and LDL-C levels. Positive effect was observed on hypolipidemic efficacy due to the regulation of PI3K/Akt/Bcl-xl pathway, as indicated by the amelioration of PI3K, Akt and Bcl-xl gene and protein expression.ConclusionThis study firstly systematically disclose the multi-ingredients, multi-targets mechanisms of mulberry leaf on obesity by using network pharmacology approach, and validate in HepG2 cells that the protective effect of MLF against obesity involved both inflammation response and lipid metabolism involving PI3K/Akt/Bcl-xl signaling pathway. It provides indications for further mechanistic research of mulberry leaf and also for the development as a potential candidate for the therapy for obese patients.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

CPT2 down-regulation promotes tumor growth and metastasis through inducing ROS/NFκB pathway in ovarian cancer

BackgroundCarnitine palmitoyltransferase 2 (CPT2) is a rate-limiting enzyme involved in fatty acid β-oxidation (FAO) regulation. Recently, it has been increasingly recognized that lipid metabolism dysregulation is closely implicated in tumorigenesis. However, the involvement of CPT2 in the progression of cancer is still largely unclear, especially in ovarian cancer (OC).MethodsIn the present study, CPT2 expression and its clinical significance were determined in OC tissues and cells. The biological functions and molecular mechanisms of CPT2 in OC growth and metastasis were determined by in vitro and in vivo assays.FindingsWe found that CPT2 was frequently down-regulated in primary ovarian serous carcinomas, which is significantly correlated with poor survival of ovarian cancer patients. Functional experiments revealed that CPT2 inhibited OC cell growth and metastasis via suppression of G1/S cell cycle transition and epithelial to mesenchymal transition (EMT), as well as induction of cell apoptosis. Mechanistically, suppression of ROS/NFκB signaling pathway by increasing fatty acid oxidation-derived NADPH production was involved in the anti-tumorigenic functions of CPT2 in OC cells.InterpretationAltogether, our findings demonstrate that CPT2 functions as a potential tumor suppressor in OC progression. CPT2 may serve as a novel prognostic marker and therapeutic target in OC.     

Molecular models of NS3 protease variants of the Hepatitis C virus

Background  

Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.     

Carnitine palmitoyltransferase 1C: From cognition to cancer

Carnitine palmitoyltransferase 1 (CPT1) C was the last member of the CPT1 family of genes to be discovered. CPT1A and CPT1B were identified as the gate-keeper enzymes for the entry of long-chain fatty acids (as carnitine esters) into mitochondria and their further oxidation, and they show differences in their kinetics and tissue expression. Although CPT1C exhibits high sequence similarity to CPT1A and CPT1B, it is specifically expressed in neurons (a cell-type that does not use fatty acids as fuel to any major extent), it is localized in the endoplasmic reticulum of cells, and it has minimal CPT1 catalytic activity with l-carnitine and acyl-CoA esters. The lack of an easily measurable biological activity has hampered attempts to elucidate the cellular and physiological role of CPT1C but has not diminished the interest of the biomedical research community in this CPT1 isoform. The observations that CPT1C binds malonyl-CoA and long-chain acyl-CoA suggest that it is a sensor of lipid metabolism in neurons, where it appears to impact ceramide and triacylglycerol (TAG) metabolism. CPT1C global knock-out mice show a wide range of brain disorders, including impaired cognition and spatial learning, motor deficits, and a deregulation in food intake and energy homeostasis. The first disease-causing CPT1C mutation was recently described in humans, with Cpt1c being identified as the gene causing hereditary spastic paraplegia. The putative role of CPT1C in the regulation of complex-lipid metabolism is supported by the observation that it is highly expressed in certain virulent tumor cells, conferring them resistance to glucose- and oxygen-deprivation. Therefore, CPT1C may be a promising target in the treatment of cancer. Here we review the molecular, biochemical, and structural properties of CPT1C and discuss its potential roles in brain function, and cancer.     

The effect of dietary l-carnitine supplementation on cold tolerance and growth of the ornamental cichlid fish Pelvicachromis pulcher—preliminary results

The effect of dietary l-carnitine supplementation on cold tolerance, growth and survival of the ornamental cichlid Pelvicachromis pulcher was tested under laboratory conditions. Fish were reared for a period of 82 days on a diet containing different levels of added l-carnitine: 0, 500 (3.1 mmol), 1000 (6.2 mmol) and 2000 (12.4 mmol) mg/kg. At the end of the growth period the fish were exposed to a cold shock. Fish which received dietary l-carnitine supplementation (at all tested levels) exhibited significantly better survival rates following exposure to a cold shock, and readapted quicker to normal temperatures compared to the control group which had no l-carnitine added to their diet. The addition of l-carnitine to the diet at a level of 1000 mg/kg seems to yield the best protection against exposure to cold shock.Growth differences among the treated fish were not significant, although the fish which received 500 mg/kg supplementation exhibited slightly better growth.     

Network pharmacology combined with metabolomics approach to investigate the protective role and detoxification mechanism of Yunnan Baiyao formulation

BackgroundYunnan Baiyao (YNBY) is a traditional Chinese medicine formulae, which has the functions of hemostasis, activating blood circulation and removing blood stasis, anti-inflammation, etc. Although the presence of Caowu (CW, Aconiti Kusnezoffii Radix), the detoxification mechanism of YNBY is still unclear.PurposeIn current study, network pharmacology, toxicological methods and metabolomics technique were applied to explore YNBY in attenuating toxicity of CW.MethodsPrediction of targets and pathways of CW were carried out by commonly used network pharmacological method. Simultaneously, SD rats were orally administrated with CW, processed CW (ZCW), YNBY, and YNBY which lack of CW (QCW) for 15 days. Tissue samples were observed with histopathology. Urine samples were analyzed with ultra-performance liquid chromatography-mass spectrometry to screen differential metabolites and related metabolic pathways associated with toxicity of CW. Furthermore, by comparing the changes of the metabolite contents, focused the attenuated metabolic pathway. Finally, the network pharmacological and experimental data were integrated to investigate detoxification mechanism of YNBY.ResultsA total of 44 potential toxicity biomarkers were identified and 14 related pathways were involved in the toxicity of CW. Furthermore, 5 core toxicity biomarkers (2-keto-6-acetamidocaproate, γ-glutamylleucine, prostaglandin E₃, 4-hydroxy-5-(3’-hydroxyphenyl)-valeric acid-3’-O-sulphate, and 3,4-dihydroxy- phenylglycol O-sulfate) were regulated to normal condition in YNBY group. Lysine degradation was locked as the core metabolic pathway of detoxification of YNBY. Integrating the predicted results of network pharmacology, ACHE, SLC6A3, SLC6A4 might be the target of protective role of other herbs in YNBY.ConclusionNetwork pharmacology combined with metabolomics exhibited a powerful mean to investigate the herbal toxicity and probed into the detoxification mechanism of formulae, which contributes to its safety evaluation.     

15-Deoxy-Δ12,14-prostaglandin J2 prevents oxidative injury by upregulating the expression of aldose reductase in vascular smooth muscle cells

Abstract

The omega-6 fatty acid derivative 15-Deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is believed to play a role in cellular protection against oxidative stress in diverse cell systems. However, the cellular mechanisms by which protection is afforded by 15d-PGJ₂ are not fully elucidated in vascular smooth muscle cells (VSMCs). In this study, we report the finding that 15d-PGJ₂ elicited a time and concentration- dependent increase in aldose reductase (AR) expression. This induction was independent of the activation of peroxisome proliferator- activated receptor γ. Inhibition of phosphatidylinositol 3-kinase (PI3K) significantly suppressed the increase in expression and promoter activity of AR induced by 15d-PGJ₂. Luciferase reporter assays demonstrated that 15d-PGJ₂ targets the multiple stress response regions comprising the antioxidant response element in the promoter of the AR gene. 15d-PGJ₂-mediated induction of AR promoter activity was potentiated in the presence of nuclear factor-erythroid 2-related factor 2 (Nrf2), but not in cells expressing dominant negative Nrf2. Cells treated with 15d-PGJ₂ were resistant to oxidant-induced apoptotic cell death by inhibiting production of reactive oxygen species. These effects were significantly attenuated in the presence of an AR inhibitor or small interfering RNA against AR, indicating that AR plays a protective role against oxidative injury. Taken together, these findings demonstrate that activation of PI3K by 15d-PGJ₂ increases the expression of AR through Nrf2, and increased AR activity may function as an important cellular response against oxidative injury.     

A regenerative approach for bone repair in congenital pseudarthrosis of the tibia associated or not associated with type 1 neurofibromatosis: correlation between laboratory findings and clinical outcome

Background and aimsCongenital pseudarthrosis of the tibia (CPT) is a rare orthopedic disease presenting spontaneous fractures that do not heal. The treatment of CPT is characterized by repeated surgical procedures that often fail, with the inevitable outcome of severe disability and amputation. We tested the hypothesis that CPT may benefit from regenerative strategies based on mesenchymal stromal cells (MSC) combined with platelet-rich fibrin (PRF) as a source of growth factors. The aim of the study was to verify whether laboratory testing to assess the osteogenic properties of MSC and the osteo-inductive activity of PRF correlated with the clinical outcome.MethodsTen patients affected by refractory CPT were treated by using MSC derived from the iliac crest (IC-MSC), PRF and lyophilized bone. In six patients, CPT was associated with type 1 neurofibromatosis (NF1). Biochemical, functional and molecular assays were performed to assess the intrinsic osteogenic potential of IC-MSC (cells cultured with fetal calf serum) and the osteo-inductive properties of PRF (cells cultured with autologous serum).ResultsBone consolidation was obtained in three patients who had CPT and NF1. In these patients, the IC-MSC exposed to autologous serum were able to form mineral nodules in vitro, while the mineralizing ability was totally abrogated in patients with a poor clinical outcome.ConclusionsCell therapy may be a useful tool for the treatment of refractory CPT because it increases the opportunity to achieve effective bone tissue regeneration. Our data suggest that the presence of pro-osteogenic growth factors is an essential requirement for bone healing.